This invention relates to a pharmaceutical composition for prophylaxis and/or treatment of apoptosis-related disease which comprises as an effective ingredient batroxobin.
Apoptosis is one of the courses or manners of cell death proposed by Kerr, Wyllie, Currie et al. (see Brit. J. Cancer, 26, 239 (1972)). In embryology, there has been known cell death which occurs infallibly at a certain stage of embryogenesis at a certain place. This is also called xe2x80x9cprogrammed cell deathxe2x80x9d to mean that it occurs according to the program of embryogenesis. Accordingly, it is morphologically differentiated from xe2x80x9cnecrosisxe2x80x9d which is the course wherein necessary cells are injured to death. Morphological features of apoptosis include lack of contact with neighboring cells, concentration of cytoplasm, chromatin condensation and nucleus condensation which relate to endonuclease activity, and nucleus segmentation. Further, there are also observed disappearance of microvilli on the cell surface and planing of the cell surface (membrane blebbing). Moreover, fragmentation of DNA by endonuclease activity is also observed and cells form cellular fragments called apoptotic body, the resultant apoptotic body is rapidly and phagocytotically degraded by neighboring cells and macrophages. As a result, it is believed that apoptosis occurs.
It has been known that living bodies keep homeostasis by balancing cell growth with cell death. In the past, regulation of cell growth has been studied well but regulation of cell death has hardly been known. It has been known that apoptosis is induced by the lack of biologically active substances such as nerve growth factor (NGF) and colony stimulating factor (CSF), apoptosis inducing factor such as tumor necrosis factor (TNF), lymphotoxin, and gene products such as c-myc and p-53 (see Cell., 69, 119 (1992); Nature, 362,849 (1993)). It has also been known that apoptosis is inhibited by apoptosis inhibiting factor such as bcl-2 (see Nature, 359, 552 (1992); Nature, 359, 554 (1992)).
Recently, it has been recognized that apoptosis has important relation to various diseases and many trials have been made to induce or regulate cell apoptosis so as to diagnose, prevent and treat these diseases, to which attention has been drawn (see Science, 267,1456 (1995)).
Further, attention has also been drawn to the relation of apoptosis with hippocampal tardive nerve cell death in postischemic nerve cell death. Namely, when both side carotid arteries of Mongolian gerbil or rat are occluded for a short time (e.g., about 10 minutes) and then recanalized, cell death is observed in the both hippocampal CA1 region after two days (48 hours) and the cells are reduced after four days, (96 hours) and vacuole is observed in dendrite, and disappear after one week.
The brain is a tissue in which oxygen consumption are highest in all the tissues and a place where a large amount of energy is consumed. Accordingly, the brain is weak against ischemia and is liable to undergo function disorder. Nerve cell death is markedly observed during embryogenesis and the cells constantly die after they are born. It is believed that about 100,000 nerve cells in human cerebral cortex will die a day. Nerve cells cannot regenerate. Accordingly, if the cells die to excess due to certain damage, function disorder will occur. Particularly, nerve cell death caused by ischemia, drugs, stress, or viruses is problematic. It becomes therefore much more important to elucidate the mechanism of the cell death so as to develop agents or methods for the treatment of neuronosis such as cerebral ischemia disorder or neuropathy due to AIDS, or to obtain a key to understand the long viability of the nerve cells.
Attention has also been drawn to the relation of apoptosis with neuro degenerative diseases such as Alzheimer""s disease or Parkinson""s disease, and it becomes more important to elucidate the mechanism of development of the diseases and to establish therapeutic methods or drugs for the treatment thereof.
An object of the present invention is to provide a pharmaceutical composition which regulates apoptosis so as to prevent and/or treat apoptosis-related diseases.
The inventors of the present invention has found that batroxobin is effective for prophylaxis and/or treatment of heart disease and cerebral disease in ischemia reperfusion and based on the finding has filed a patent application (U.S. Ser. No. 08/665,982 filed Jun. 19, 1996 claiming the priority of Japanese Patent Application No. Hei 7-161665). The invention is based on the finding that batroxobin inhibits xe2x80x9cnecrosisxe2x80x9d which is a course in which cells are damaged to death. As described earlier, xe2x80x9capoptosisxe2x80x9d and xe2x80x9cnecrosisxe2x80x9d are morphologically different and the application is silent with respect to the relation between batroxobin and inhibition of apoptosis. The inventors of the present invention has studied the pharmacological action of batroxobin more in detail and found that batroxobin has the action to inhibit apoptosis and accomplished the present invention based on this finding.
Batroxobin used in the present invention is a thrombin-like enzyme derived from snake venom of Bothrops atrox moojeni and examples of commercially available formulations of the enzyme include xe2x80x9cbatroxobin formulationxe2x80x9d manufactured by Tobishi Pharmaceutical Co., Ltd.
The pharmaceutical composition for prophylaxis and/or treatment of apoptosis-related diseases of the present invention inhibits apoptosis and can be used as a drug for prophylaxis and/or treatment of apoptosis-related diseases. Examples of the apoptosis-related diseases include ischemic disease (excluding reperfusion injury), neuro degenerative disease, peripheral nerve damage, apalstic anemia, liver damage and HIV infection. Examples of the ischemic diseases include heart disease such as cardiac infarction, cardiac angina, congestive heart failure and arrhythmia, and cerebrovascular diseases such as cerebral stroke, subarachnoidal hemorrhage, cerebral infarction, and cerebral thrombosis. Examples of the neuro degenerative diseases include Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, pigmented retinitis, and cerebelli degeneration.
Batroxobin formulation (a thrombin-like enzyme derived from snake venom of Bothrops atrox moojeni) (1 ml) contains the following components.
Dosage of batroxobin in the present invention depends on the conditions and typically 1 to 20 batroxobin units (Batroxobin Unit, abbreviated as BU) per day per one time for human adult although it may vary depending on the conditions. The batroxobin formulation can suitably diluted and administered by intravenous drip infusion, intravenous injection or intraarterial injection. The batroxobin unit described herein is a unit representing an enzymatic activity of batroxobin and such an activity that the coagulation of plasma is taken place in 19.0xc2x10.2 seconds when 0.1 ml of a batroxobin solution is added to 0.3 ml of standard human plasma containing citric acid at a temperature of 37xc2x0 C. is defined as 2 BU.
Acute toxicity test for batroxobin was conducted by intravenous administration to mice, rats, rabbits and dogs. The resulted LD50 values (BU/kg) were as follows: